Use or cyclolignans and new cyclolignans

ABSTRACT

The invention refers to new compounds and the use thereof as well as the use of known cyclolignans having a trans configuration of the lactone ring as inhibitors of the insulin-like growth factor-1 receptor. Said compounds can be used for treatment of IGF-1R dependent diseases, especially cancer. Especially deoxypodophyllotoxin can be used for treatment of leukemia in humans.

[0001] The present invention refers to new compounds as well as to theuse of new and known compounds inhibiting the insulin-like growthfactor-1 receptor, the IGF-1R, for treatment of IGF-1R dependentdiseases, especially cancer.

BACKGROUND OF THE INVENTION

[0002] The insulin-like growth factor-1 receptor (IGF-1R) plays animportant role in proliferation, protection against apoptosis andtransformation of malignant cells. The IGF-1R is also important formaintaining the malignant phenotype of tumour cells, and is involved intumour cells developing resistance to the action of anti-cancer drugs.In contrast, the IGF-1R seems not to be an absolute requirement fornormal cell growth.

[0003] The IGF-1R consists of two identical extracellular alpha-subunitsthat are responsible for ligand binding, and two identical beta-subunitswith a transmembrane domain and an intracellular tyrosine kinase domain.The ligand-receptor interaction results in phosphorylation of tyrosineresidues in the tyrosine kinase domain, which spans from amino acid 973to 1229 of the β-subunit. The major sites for phosphorylation are theclustered tyrosines at position 1131, 1135 and 1136 (LeRoith, D., etal., Endocr Rev 1995 April; 16(2), 143-63). After autophosphorylation,the receptor kinase phosphorylates intracellular proteins, like insulinreceptor substrate-1 and Shc, which activate the phosphatidyl inositol-3kinase and the mitogen-activated protein kinase signalling pathways,respectively.

[0004] Based on the pivotal role of IGF-1R in malignant cells, itbecomes more and more evident that IGF-1R is a target for cancer therapy(Baserga, R., et al., Endocrine vol. 7, no. 1, 99-102, August 1997). Onestrategy to block IGF-1R activity is to induce selective inhibition ofthe IGF-1R tyrosine kinase. However, today there are no selectiveinhibitors of IGF-1R available.

[0005] Drugs containing the cyclolignan podophyllotoxin has been usedsince centuries, and its anti-cancer properties have attractedparticular interest. Undesired side effects of podophyllotoxin have,however, prevented its use as an anti-cancer drug. The mechanism for thecytotoxicity of podophyllotoxin has been attributed to its binding tobeta-tubulin, leading to inhibition of microtubule assembly and mitoticarrest. The effect of podophyllotoxin on microtubules required μMconcentrations in cell free systems. The trans configuration in thelactone ring of podophyllotoxin has been shown to be required forbinding to beta-tubulin. In agreement with this, its stereoisomerpicropodophyllin, which has a cis configuration in the lactone ring, hasa 50-fold lower affinity for microtubuli and a more than 35-fold higherLD50 in rats. Because of the low affinity for microtubuli ofpicropodophyllotoxin this compound has attracted little interest. Duringthe last decades the major interest on podophyllotoxin derivatives hasconcerned etoposide, which is a ethylidene glucoside derivative of4′-demethyl-epipodophyllotoxin. Etoposide, which has no effect onmicrotubules, is a DNA topoisomerase II inhibitor, and is currentlybeing used as such in cancer therapy. A 4′-hydroxy instead of a4′-methoxy group of such cyclolignans is an absolute requirement forthem to inhibit topoisomerase II.

PRIOR ART

[0006] A number of synthetic tyrosine kinase inhibitors, calledtyrphostins, have been studied by Parrizas, M., et al., Endocrinology1997, Vol. 138, No. 4, 1427-1433. The IGF-1R is a member of the tyrosinekinase receptor family, which also includes the receptors of insulin,epidermal growth factor (EGF), nerve growth factor (NGF), andplatelet-derived growth factor (PDGF). All of the tyrphostins active onIGF-1R cross-react with the insulin receptor, since they are highlyhomologous, although two of the tyrphostins showed a moderate preferencefor IGF-1R. It was therefore suggested that it could be possible todesign and synthesize small molecules capable of discriminating betweenthese two receptors.

[0007] Substrate competitive inhibitors of IGF-1 receptor kinase arediscussed by Blum, G., et al. in Biochemistry 2000, 39, 15705-15712. Anumber of lead compounds for inhibitors of the isolated IGF-1R kinaseare reported. The search for these compounds was aided by the knowledgeof the three-dimensional structure of the insulin receptor kinasedomain, which is 84% homologous to the IGF-1R kinase domain. The mostpotent inhibitor found was tyrphostin AG 538, with an IC50 of 400 nM.However, said inhibitor also blocked the insulin receptor kinase.

[0008] Kanter-Lewensohn, L., et al., Molecular and CellularEndocrinology 165 (2000), 131-137, investigated whether the cytotoxiceffect of tamoxifen (TAM) on melanoma cells could depend on interferencewith the expression or function of the insulin-like growth factor-1receptor. It was found that, although TAM did not have a strong effecton IGF-1 binding and the expression of IGF-1R at the cell surface, at 15microM TAM efficiently blocked tyrosine phosphorylation of the IGF-1Rbeta-subunit.

[0009] A connection between the IGF-1R and podophyllotoxin derivativeshas never previously been made. The Chemistry of Podophyllum by J. L.Hartwell et al., Fortschritte der Chemie organischer Naturstoffe 15,1958, 83-166, gives an overview of podophyllotoxin and differentderivatives thereof, commercially derived from two species of plants,Podophyllum peltatum and Podophyllum emodi. As said, the observedcytotoxic effect of podophyllotoxin has been ascribed to its binding tomicrotubuli resulting in a mitotic block. The same effects on cells havebeen described for deoxypodophyllotoxin and this was suggested to be thereason why these two compounds and their corresponding 4′-demethylanalogues could be used for treatment of psoriasis (WO 86/04062).However, whereas the LD50 of podophyllotoxin in rats is relatively low(14 mg/kg), LD50 of the deoxy derivative is strangely >15-fold higher.Other podophyllotoxin derivatives, for which LD50 in rats was high, suchas acetylpodophyllotoxin (185 mg/kg) and epipodophyllotoxin (>200 mg/kg)have been considered to essentially lack biological activity(Seidlova-Masinova V., et al. J Nat Cancer Inst, 18, 359-371, 1957).

[0010] Structure-activity evaluation of a number of morpholinoderivatives of benzyl-benzodioxole having a structural similarity topodophyllotoxin were performed by Batra, J., et al., BiochemicalPharmacology, Vol. 35, No. 22, 4013-4018, 1986. The ability of thecompounds to inhibit tubulin polymerisation was tested, but themorpholino compound most similar to podophyllotoxin was the least activein the series.

[0011] Benzyl and cinnamylderivatives of 2,4-di-tert-butylphenol and of1,3-benzodioxoles are known as insect chemosterilants from Jurd, L., etal., J. Agric. Food Chem. Vol. 27, No. 5, 1007-1016, 1979.

OBJECTS OF THE INVENTION

[0012] The object of the invention is to find new compounds and newmethods for treatment of IGF-1R dependent diseases, especially cancer,by means of an inhibition of the insulin-like growth factor-1 receptor.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 shows a computer model of the 12 amino acid peptidecomprising the tyrosines 1131, 1135 an 1136 of the IGF-1 receptor.

[0014]FIG. 2A shows the structural formulas of the compoundspodophyllotoxin, deoxypodophyllotoxin, and FIG. 2B shows the structuralformulas of epipodophyllotoxin and acetylpodophyllo-toxin.

DESCRIPTION OF THE INVENTION

[0015] The three-dimensional structure of short peptides having theamino acid sequence of the IGF-1R tyrosine domain, including thetyrosine residues at position 1131, 1135 an 1136, were analysed using acomputer programme in order to find compounds having the ability tomimick the tyrosine residues and interfere with their phosphorylation.It was then discovered when using a 12-amino acid peptide that two ofthe three key tyrosines, that is 1135 and 1136, which have to beautophosphorylated in IGF-1R for activation, could be situated as closeas 0.95 nm (9.5 Å) from each other, and that the apparent angle betweenthese groups was about 60°. The configuration of said sequence is shownin FIG. 1. Such a short distance has not been observed for thecorresponding tyrosines in the insulin receptor. FIG. 1 also depicts thespace structures of podophyllotoxin and deoxypodophyllotoxin.

[0016] Molecular modelling showed that an inhibitory molecule couldconsist of two benzene rings separated by only one carbon atom. When atwo-carbon bridge was tried, the distance between the substituents ofthe benzene rings was too long, about 1.3 nm (13 Å).

[0017] The substituents of the inhibitors corresponding to the hydroxygroups in the tyrosines were selected to be methoxy or methylenedioxygroups, since they are chemically relatively stable, i.e. they are notoxidized or phosphorylated. The distance between these substituentsshould be about 0.95±0.10 nm (9.5±1.0 Å).

[0018] It was then surprisingly found that the two angled benzene ringsof some cyclolignans, including podophyllotoxin, could mimick almostexactly the two tyrosines 1135 and 1136, indicating that podophyllotoxinand derivatives could interfere with the autophosphorylation of thesetyrosine residues.

[0019] In order to penetrate the receptor, an inhibitory molecule has tobe small. When for instance podophyllotoxin was conjugated with aglucoside derivative,podophyllotoxin-4,6-O-benzylidene-β-D-glucopyranoside, the effect onIGF-1R completely disappeared. Furthermore, following reduction of thelactone ring to a diol structure, the size of the molecule increased dueto the reduced substituents sticking out from the molecule, resulting ina dramatically reduced activity of the compounds. Increasing the size byforming methylenedioxy derivatives or acetonides of podophyllotoxindiolalso resulted in compounds with little or no activity.

[0020] The inhibitor molecule also has to be relatively nonpolar, sothat it can freely penetrate cell membranes and the IGF-1 receptor, butsufficiently polar to be reasonably soluble in water. The polarity ofthe molecule is determined by the number and nature of the oxygenfunctions. The polarity seems to be optimal when the water solubility isbetween that of deoxypodophyllotoxin, i.e. about 0.01 mM, and that ofpodophyllotoxin, about 0.1-0.2 mM. No charged or highly polar groupsshould be present on the molecule.

[0021] The invention refers to the use of a compound comprising theformula

[0022] wherein the distance between the carbon atom of the methylenegroup and the carbon atom of the methoxy group is 0.85-1.05 nm;

[0023] R is OH, OCH₃, OC₂H₅ or a C1-5 linear or branched hydrocarbonchain, optionally having 1-3 oxygen functions, and optionally forming abond with the carbon (number 5) in the top benzene ring.

[0024] R₁, which can be the same or different, is OH or OCH₃, and n is0-2;

[0025] as an inhibitor of tyrosine phosphorylation of the insulin-likegrowth factor-1 receptor.

[0026] Oxygen functions in this context refer to hydroxy, oxo, carboxy,methoxy, methylenedioxy, lactone, ether and/or ester groups.

[0027] One group of compounds which can be used in accordance with theinvention has the formula Ib

[0028] wherein R is OH, OCH₃, OC₂H₅,CH₃, C₂H₅, or C₂H₄OH; and R₁ and nare as defined above. The substituent R of the compounds of this groupcan be in the R or S position in relation to the bottom benzene ring.

[0029] Compounds of the formula I can be prepared by the followingrepresentative synthesises:

[0030] As examples of compounds which can be prepared in this way can bementioned:.

[0031] 6-(3,4,5-trimethoxy-alpha-hydroxy-benzyl)--1,3-benzodioxole (Ia)

[0032] 6-(3,4,5-trimethoxy-alpha-methoxy-benzyl)-1,3-benzodioxole (Ib)

[0033] 6-(3,4,5-trimethoxy-alpha-methyl-benzyl)-1,3-benzodioxole (Ic)

[0034] Another group of compounds which can be used in accordance withthe invention has the formula II

[0035] wherein R₂, R₃ and R₄, which can be the same or different, are H,OH, O, OCH₃, OC₂H₅ or R₂ and R₃ together is a methylenedioxy group, orR₃ and R₄ together is an acetonide, carbonate or methylendioxy group;and R₁ and n are as defined above. The substituents R₂, R₃ and R₄ can,when not expressing an oxo group, be in either alpha- or beta-position.The bottom benzene ring should preferably be in the alpha-position.

[0036] Compounds of the formula II can be prepared by the followingrepresentative synthesises:

[0037] In said schedules NBS is N-bromosuccinimide, PPA ispolyphosphoric acid, TTN is thalliumnitrate trihydrate, p-TSA isp-toluene sulfonic acid, DMF is N,N-dimethylformamide, and LDA islithium dialkylamide.

[0038] As examples of compounds which can be prepared in this way can bementioned:

[0039] 1-(4-methoxy-phenyl)-6,7-methylenedioxy-1,2,3,4,-tetrahydronaphtalene (II a)

[0040]3-hydroxy-l-(4-methoxy-phenyl)-6,7-methylenedioxy-1,2,3,4-tetrahydronaphtalene(II b)

[0041] 1-(3,4,5-trimethoxy-phenyl)-6,7-methylenedioxy-1,2,3,4,-tetrahydronaphtalene (II g)

[0042]3-hydroxy-l-(3,4,5-trimethoxy-phenyl)-6,7-methylenedioxy-1,2,3,4-tetrahydronaphtalene(II h).

[0043] Compounds of the formula II can also be prepared by the followingsynthesis:

[0044] As examples of-compounds which can be prepared in this way can bementioned:

[0045]2-hydroxy-1-(4-methoxy-phenyl)-6,7-methylenedioxy-1,2,3,4-tetrahydronaphtalene(II c)

[0046]1-(4-methoxy-phenyl)-2-oxo-6,7-methylenedioxy-1,2,3,4-tetrahydronaphtalene(II d)

[0047]2-hydroxy-1-(3,4,5-trimethoxy-phenyl)-6,7-methylenedioxy-1,2,3,4-tetrahydronaphtalene(II i)

[0048]1-(3,4,5-trimethoxy-phenyl)-2-oxo-6,7-methylenedioxy-1,2,3,4-tetrahydronaphtalene(II j).

[0049] Compounds of the formula II can also be prepared as follows:

[0050] As examples of compounds which can be prepared in this way can bementioned:

[0051] 2, 3-dihydroxy-1-(4-methoxy-phenyl)-6, 7-methylenedioxy-1,2,3,4-tetrahydronaphtalene (II e)

[0052]1-(4-methoxy-phenyl)-2,3-methylenedioxy-6,7-methylenedioxy-1,2,3,4-tetrahydronaphtalene(II f)

[0053]2,3-dihydroxy-1-(3,4,5-trimethoxy-phenyl)-6,7-methylenedioxy-1,2,3,4-tetrahydronaphtalene(II k)

[0054]1-(3,4,5-trimethoxy-phenyl)-2,3-methylenedioxy-6,7-methylenedioxy-1,2,3,4-tetrahydronaphtalene(II l)

[0055] Details of the reaction conditions in the above synthesises aredescribed in Advanced Organic Chemistry, Jerry March (ed.), 4^(th)edition, Wiley-Interscience Publication, New York, 1992.

[0056] Still another group of compounds which can be used in accordancewith the invention are compounds of the formula III

[0057] wherein R₂, R₅, R₆, which can be the same or different, are H,OH, OOCH₃, OOCH₂CH₃, OCH₃, or OC₂H₅, or R₅ and R₆ together is an etheror a lactone; and R₁ and n are as defined above. The substituent R₂,when being a free hydroxy group cannot be in the alpha-position, as inpodophyllotoxin. Other R₂ substituents, except an oxo group, can be ineither alpha- or beta-position. Notably, the bottom benzene ring is inthe alpha-position and there is a beta-bond between the carbons numbered8 and 9 and an alpha-bond between the carbons numbered 8′ and 9′, thusthey form a trans configuration, as in for example deoxypodophyllotoxinand podophyllotoxin.

[0058] The invention especially refers to the use of any of therelatively non-toxic cyclolignans, such as epipodophyllotoxin,deoxypodophyllotoxin and acetylpodophyllotoxin, as an inhibitor oftyrosine autophosphorylation of the insulin-like growth factor-1receptor, whereas the use of more cytotoxic and tissue irritatingcompounds, such as podophyllotoxin and 4′-demethyl-podophyllotoxin,should be avoided.

[0059] Some compounds of the formula III are naturally occurring inplants, such as deoxypodophyllotoxin and podophyllotoxin. For thepreparation of said substances in pure form, dried and finely groundrhizomes of e.g. Podophyllum emodi or Podophyllum peltatum are extractedwith organic solvents. The extract is then filtered and concentrated onsilica gel. The fractions containing the substances are collected andthe latter are further purified by chromatography on acid alumina andsilica gel etc., and finally recrystallized.Podophyllotoxin may be usedas the starting material for the syntheses of its less toxicderivatives.

[0060] Epipodophyllotoxin is readily prepared from podophyllotoxin. Fivemg of the latter are dissoved in 2.5 mL of acetone. To the solution isadded 0,5 mL of concentrated HCl, and the mixture is boiled for 2 hours.The solution is then neutralized with aqueous NaHCO3 (about 0.5 g in 5mL) and following evaporation of the acetone, the productepipodophyllotoxin is extracted with ethyl acetate.

[0061] Acetylpodophyllotoxine (the acetate derivative ofpodophyllotoxin) can be prepared from podophyllotoxin by incubating 0.1mg of the latter with 1 mL of acetic anhydride and 1 mL of pyridine at50° C. for 16 hours. The reagents are then partly evaporated, 10 mL ofwater and 10 mL of ethyl acetate are added and the product is thenextracted from the aqueous phase.

[0062] Acetonides and methylenedioxy derivatives can be preparedstarting from diols obtained by reducing the lactone ring of naturallignans according to standard procedures.

[0063] As additional examples of compounds of the formula III can bementioned: podophyllotoxone, and 4′-demethyl-deoxypodophyllotoxin.

[0064] The invention also refers to the new compounds of the formula I

[0065] wherein R is OH, OCH₃, OC₂H₅, CH₃, C₂H₅, or C₂H₄OH; and R₁ and nare as defined above, with the proviso that when R₁ is OCH₃ and n is 2,R is not OH, that when R is a CH₃ or C₂H₅, R₁ is OH and n is 1 or 2,that when R is C₂H₅, R₁ is not OH.

[0066] The molecule of the invention should be relatively rigid, inorder to keep the distance between the two substituents within the givenrange, that is 0.95±0.10 nm (9.5±1.0 Å). Forming a ring structure of thehydrocarbon chain will prevent rotation or motion of the benzene ring,and so does lactone formation.

[0067] The invention also refers to the new compounds of the formula II

[0068] wherein R₂, R₃ and R₄, which can be the same or different, are H,OH, O, OCH₃, or R₂ and R₃ together is a methylenedioxy group, or R₃ andR₄ together is an acetonide, carbonate or methylendioxy group; and R₁and n are as defined above.

[0069] To design an inhibitor of the IGF-1R tyrosine kinase fortherapeutic purposes it is of critical importance that the inhibitordoes not cross-react with the insulin receptor kinase, which is highlyhomologous to the IGF-1R. Co-inhibition of the insulin receptor willlead to a diabetogenic response in-vivo. This response comprises a veryserious side effect, which cannot be overcome by insulin treatment sincethe receptor kinase is being blocked. We have demonstrated thatpodophyllotoxin and derivatives, which are much more potent IGF-1Rinhibitors than the tyrophostin-based compounds, do not interfere withthe insulin receptor tyrosine kinase at all. Neither do they interferewith tyrosine phosphorylation of the receptors of epidermal growthfactor, platelet-derived growth factor or fibroblast growth factor.

[0070] Podophyllotoxin has for long been implicated in cancer therapy,but in the way it was administered to patients it produced unacceptableside effects. The anti-cancer effect, as well as the side effects, wasattributed to inhibition of microtubule assembly and mitotic block. Ithas now been demonstrated that podophyllotoxin and some of its lesstoxic analogues are very potent and specific inhibitors of tyrosinephosphorylation of the insulin-like growth factor-1 receptor, whichplays a pivotal role as a survival factor in cancer cells. Compared tothe anti-microtubule effect of podophyllotoxin, a 100-fold lowerconcentrations were sufficient to inactivate the IGF-1R. Of utmostimportance is that podophyllotoxin and analogues do not inhibit theinsulin receptor, which is highly homologous to IGF-1R. Moreover, theydo not inhibit other major growth factor receptor kinases either.

[0071] Relatively nontoxic compounds of the formula I can be used fortreatment of IGF-1R dependent diseases, such as cancer,arteriosclerosis, including prevention of restenosis of the coronaryarteries after vascular surgery, psoriasis and acromegaly.

[0072] A pharmaceutical composition comprising a compound of the formulaI in combination with a physiologically acceptable carrier and optionaladditives can be administered to a patient by any suitable route, suchas parenterally, preferably by intravenous infusion, or topically, forinstance by a patch.

[0073] The invention refers to the new compounds of the formula I or IIfor use as a medicament, and especially for the preparation of amedicament for treatment of cancer.

[0074] The results of the biological experiments suggest thatsubmicromolar concentrations of podophyllotoxin, or of less toxicanalogues such as deoxypodophyllotoxin or epipodophyllotoxin, can besufficient to cause tumour cell death. However, it is believed that itis important to keep a constant plasma concentration of the inhibitorsover lengthy periods, to allow them to continuously saturate allIGF-1Rs, and in this way eventually kill as many malignant cells aspossible. Therefore, continous infusion of podophyllotoxin derivatives,in connection with monitoring the plasma concentration, may be thestrategy of treatment instead of repetetive (e.g. daily) injections,which may lead to repeated reactivations of IGF-1R between thetreatments.

[0075] The invention consequently also refers to a method of treatmentof a cancer in a mammal, comprising the steps of administrating apharmaceutical composition, containing a compound having the formula Iin combination with a physiologically acceptable carrier, by constantinfusion to a patient suffering from a tumour, controlling the plasmalevel of the compound, and adjusting the rate of infusion to keep theplasma level between 0.05 and 5.0 μM (depending on the general toxicityof the copound), for a period of time being sufficient for the tumour tobe retarded or to disappear.

[0076] In case of tumours not completely dependent on IGF-1R, thecompounds of the invention can be useful to sensitise the tumour cellsto the effects of other anti-cancer drugs.

EXPERIMENTAL Materials

[0077] Chemicals

[0078] Cell culture reagents, that is media, fetal calf serum andantibiotics, were purchased from Gibco, Sweden. All other chemicalsunless stated otherwise were from Sigma (St. Louis. Mo., USA). A mousemonoclonal antibody against phosphotyrosine (PY99) and a polyclonalantibody against α-subunit of IGF-1R (N20) were obtained from Santa CruzBiotechnology Inc (Santa Cruz, Calif., USA). A monoclonal antibodyagainst the α-subunit of IGF-1R (IR-3) was purchased from OncogeneScience (N.Y., USA). Deoxypodophyllotoxin and podophyllotoxin (99.97%purity), and acetylpodophyllotoxin, podophyllotoxone and4′-demethylpodophyllotoxin (>95% purity) were obtained as gifts fromAnalytecon SA, Switzerland.

[0079] Cell Cultures

[0080] The human malignant melanoma cell lines SK-MEL-2, SK-MEL-5 andSK-MEL-28, the prostatic carcinoma cell line PC-3, and the breast cancercell line MCF-7 were from the American Tissue Culture Collection, USA.The malignant melanoma cell lines BE, and FM55 were obtained fromProfessor R Kiessling, CCK, Karolinska Hospital, Stockholm, Sweden. TheR- and P6 cell lines were gifts from Professor R. Baserga, ThomasJefferson University, Philadelphia, Pa., USA. All cell lines werecultured in Minimal Essential Medium containing 10% faetal bovine serum,glutamine, 1% benzylpenicillin and streptomycin. The cells were grown inmonolayers in tissue culture flasks maintained at 95% air/5% CO₂atmosphere at 37° C. in a humidified incubator. For the experimentscells were cultured in either 35-mm or 60-mm plastic dishes or 96-wellplastic plates. The experiments were initiated under subconfluent growthconditions.

[0081] The human chronic myeloid leukemia K562/S and K562/Vcr30 linesand the acute myeloid leukemia cell lines HL60/0 and HL60/Nov wereobtained from ATCC. The K562/S and HL60/0 are wild type (non-resistant)cells, whereas K562/Vcr30 and HL60/Nov are cytostatic-resistantsublines. All leukemia cell lines were cultured in RPMI 1640 mediumsupplemented with 10% fetal bovine serum and with 2 mM glutamine, 1%benzyl-penicillin and streptomycin. The cells were grown in tissueculture flasks maintained at 95% air/5% CO₂ atmosphere at 37° C. in ahumidified incubator. For the experiments 25,000 cells were cultured in60-mm plastic dishes or 96-well plastic plates. The experiments onleukemia cells were performed in collaboration with Associate professorSigurd Vitols, Department of Pharmacology, Karolinska Hospital(Stockholm, Sweden).

[0082] Methods

Assay of Cell Growth and Survival

[0083] Cell proliferation kit II (Roche Inc.) is based on colorimetricchange of the yellow tetrazolium salt XTT in orange formazan dye by therespiratory chain of viable cells (Roehm, N W, et al., J Immunol Methods142:257-265, 1991). Cells seeded at a concentration of 5000/well in 100μl medium in a 96-well plate were treated with different drugs in thegiven concentration. After 24 or 48 h the cells were incubated,according to the manufacturer 's protocol, with XTT labelling mixture.After 4 h the formazan dye is quantified using a scanning multiwellspectrophotometer with a 495-nm filter. The absorbance is directlycorrelated with number of viable cells. The standard absorbance curvewas drawn by means of untreated cells seeded at a concentration of from1000 to 10 000 cells/well with an increasing rate of 1000 cells/well.All standards and experiments were performed in triplicates.

Immunoprecipitation and Determination of Protein Content

[0084] The isolated cells were lyzed in 10 ml ice-cold PBSTDS containingprotease inhibitors (Carlberg, M., et al., J Biol Chem 271:17453-17462,1996). 50 μl protein A or G agarose was added in 1 ml sample andincubated for 15 min at 4° C. on an orbital shaker. After centrifugationfor 10 min at 10,000 r/min at 4° C. the supernatant was saved. Theprotein content was determined by a dye-binding assay with a reagentpurchased from Bio-Rad. Bovine serum albumin was used as a standard. 15μl Protein G Plus agarose and 5 μl anti-IGF-1R were added. After a 3 hincubation at 4° C. on an orbital shaker the precipitate was collectedby pulse centrifugation in a micro centrifuge at 14,000×g for 10 s. Thesupernatant was discarded and the pellet was washed 3 times with PBSTDS.

Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS-PAGE)

[0085] Protein samples were solved in a 2×-sample buffer containingLaemmli buffer and 0.5% methanol and boiled for 5 min at 96° C. Sampleswere separated by SDS-PAGE with a 4% stacking gel and 7.5% separationgel. Molecular weight markers (Bio Rad, Sweden) were run simultaneouslyin all experiments.

[0086] Western Blotting

[0087] Following SDS-PAGE the proteins were transferred overnight tonitro-cellulose membranes (Hybond, Amersham, UK) and then blocked for 1h at room temperature in a solution of 4% skimmed milk powder and 0.02%Tween 20 in PBS, pH 7.5. Incubations with the primary antibodies wereperformed for 1 h at room temperature, followed by 3 washes with PBSwith Tween and incubation with the second antibody for 1 h roomtemperature. After another 3 washes the membranes were incubated withStreptavidin-labelled horseradish peroxidase for 30 min and thendetected using Amersham ECL system (Amersham, UK). The films werescanned by Fluor-S (BioRad).

[0088] Assay of IGF-1R Autophosphorylation in vitro

[0089] IGF-1R tyrosine autophosphorylation was analysed by a sandwichELISA assay. Briefly, 96-well plates (Immunolon, Nunc) were coatedovernight at 4° C. with 1 μg/well of the monoclonal antibody Ab-5(LabVision) to the IGF-1R beta subunit. The plates were blocked with 1%BSA in PBS Tween for 1 h, then 80 g/well of total protein lysate fromthe P6 cell line was added. As a negative control was used total proteinlysate from R-cell line. The investigated compounds were added intyrosine kinase buffer without ATP at room temperature for 30 min, priorto kinase activation with ATP. Kinase assay was performed using theSigma kit. After spectrophotometry the IC50 values of inhibitors weredetermined using the Regression function of Statistica program.

Experiment 1. Effect of Podophyllotoxin and Other Biologically ActivePhenolic Compounds on Phosphorylation of IGF-1R in Cultured MelanomaCells

[0090] Melanoma cells (line FM55) were seeded in 6-cm dishes, at aconcentration-of 10,000 cells/cm² in Minimal Essential Mediumsupplemented with lot fetal calf serum (FCS). When the cells reached aconcentration of 65,000 cells/cm², they were treated with genistein,tamoxifen, quercetin and podophyllotoxin to a final concentration of 0,1, 15 or 60 μM in the culture medium for 1 h. Treatment with 0 μMrepresents untreated controls. The cells were then isolated andsubjected to immuno-precipitation of the IGF-1R. The immunoprecipitates,containing purified IGF-1R, were fractionated by gel electrophoresis.Phosphorylation of IGF-1R was detected by an anti-phosphotyrosineantibody using Western blotting. The obtained signals representphosphorylated IGF-1R and the intensity of signals represents amounts ofphosphorylated IGF-1R. Details of the methods used are described above.The intensities are quantified by a scanner, which measures the opticaldensity (OD) of the signals. For the control cells the OD is set at100%. The blank (OD 0%) represents the background. The results given inTable 1 below are mean values of 3 experiments. TABLE 1 Level of IGF-1Rphosphorylation in intact cells Compound 1 μM 15 μM 60 μM Genistein 10096 35 Tamoxifen 95 20 10 Quercetin 100 105 96 Podophyllotoxin 8 4 2

[0091] The results show that podophyllotoxin almost completely blocksIGF-1R phosphorylation at all three concentrations, whereas genisteinonly has a partial inhibitory effect at 60 μM and quercetin has noeffect at all.

Experiment 2. Effect of Podophyllotoxin Derivatives onAutophosphorylation of IGF-1R in Cultured Melanoma Cells

[0092] FM 55 melanoma cells were cultured in the same way as describedin Experiment 1. When reaching a density of 65,000 cells/cm² in thedishes, they were treated for 1 h with 0.05 μM podophyllotoxin,deoxypodophyllotoxin, acetylpodophyllotoxin, epipodophyllotoxin,4′-demethyl-podophyllotoxin and podophyllotoxone. The cells were thenharvested for assay and quantification of IGF-1R autophosphorylation asdescribed above. The values shown in Table 2 represent means of 3experiments. TABLE 2 Inhibitory effect on IGF-1R autophosphoryl-ation inintact cells in relation to podophyllotoxin Compound Relative potencyPodophyllotoxin 1 Deoxypodophyllotoxin 0.8 Acetylpodophyllotoxin 1.3Epipodophyllotoxin 0.5 4′-demethylpodophyllotoxin 0.5 Podophyllotoxone0.3

[0093] The results show that acetylpodophyllotoxin, podophyllotoxin, anddeoxypodophyllotoxin are potent inhibitors of IGF-1R phosphorylation.

Experiment 3. Dose-Response Effects of Podophyllotoxin andDeoxypodophyllotoxin on Viability of Solid Tumour Cells

[0094] 5 different types of cell lines were seeded in 96-well plates(medium volume in a well was 100 μl), at a concentration of 10,000cells/cm² in Minimal Essential Medium supplemented with fetal calfserum. When the cells had reached a concentration of 65,000 cells/cm²,they were treated with different doses of podophyllotoxin anddeoxypodophyllotoxin for 48 h. Cell viability was then assayed (seeabove). IC50 values for each inhibitor and cell line, calculated as theconcentration, resulting in a 50% decrease in cell survival, are shownbelow. The results are based on 4 different experiments. TABLE 3 IC50(μM) for cell viability Cell line Origin PodophyllotoxinDeoxypodophyllotoxin SK-MEL-28 melanoma 0.05 0.04 BE melanoma 0.05 ndFM55 melanoma 0.04 0.04 MCF-7 Breast cancer 0.07 0.03 PC-3 prostatecancer 0.06 Nd

[0095] This shows that podophyllotoxin and deoxypodophyllotoxin are bothvery potent inhibitors of tumor cell viability.

Experiement 4. Dose-Response Effects of Different PodophyllotoxinAnalogues

[0096] FM55 melanoma cells were cultured in the same way as described inExperiment 3 and were treated with different doses of podophyllotoxinanalogues as described in Experiment 3. The results (IC50 values) aregiven in the following Table 4. TABLE 4 IC50 (μM) for viability of FM55cells Compound IC50 Podophyllotoxin 0.05 Deoxypodophyllotoxin 0.04Acetylpodophyllotoxin 0.03 4′-demethylpodophyllotoxin 0.04

[0097] The result shows that the tested analogues were all potent.

Experiment 5. Dose-Response Effects of Podophyllotoxin Analogues onViability of Leukemia Cell

[0098] The leukemia cell lines K562/S, K562/Vcr 30, HL60/0 and HL60/Novwere proven to express the IGF-1R.This was assayed by Western blottinganalysis, as described in Methods and Experiment 1 and 2. The 4 leukemiacell lines were seeded in 96-well plates (medium volume in a well was100 μl), in RPMI40 medium supplemented with fetal calf serum. After 24 hpodophyllotoxin, deoxypodophyllotoxin and other derivatives were addedat different concentrations for 72 h. Cell viability was then assayed(see above). IC50 values for each inhibitor and cell lines are shownbelow (Table 5). The results are based on 3 different experiments. TABLE5 IC50 (nM) for viability of leukemia cell lines K562/S K562/Vcr30 HL60HL60/Nov Podophyllotoxin 4 7 3 2 Deoxypodophyllotoxin 3 4 3 2Acetylpodophyllotoxin 80 140 48 46 Epipodophyllotoxin 195 450 127 90Podophyllotoxone >500 >500 >500 >500 4′-Demethyl- 22 50 20 18podophyllotoxin

[0099] The results demonstrate-that deoxypodophyllotoxin has anunexpected and exceptionally strong cytotoxic effect on human leukeimacells. Such an effect can not be explained solely by its action onIGF-1R. The effects of acetylpodophyllotoxin and epipodophyllotoxin areas expected for an IGF-1R inhibitor.

CONCLUSION

[0100] It has been demonstrated that certain cyklolignans such aspodophyllotoxin and some analogues deoxypodophyllotoxin are highlyspecific and potent inhibitors of the IGF-1R tyrosine kinase, as assayedin intact cells. When administered to intact cells the EC50 value was aslow as 0.02-0.06 μM.

[0101] Podophyllotoxin-induced inactivation of the insulin-like growthfactor-1 receptor caused extensive cell death in malignant cells,whereas cells devoid of insulin-like growth factor-1 receptors wereresistant. The non-toxic derivative picropodophyllin was equipotent topodophyllotoxin in inhibiting the insulin-like growth factor-1 receptoractivity and inducing cell death. This new mechanism of podophyllotoxinand derivatives may be useful in therapy of cancer and other IGF-1Rdependent diseases.

1. Use of a compound having the formula I

wherein the distance between the carbon atom of the methylene group andthe carbon atom of the methoxy group is 0.85-1.05 nm; R is OH, OCH₃,OC₂H₅ or a C₁₋₅ linear or branched hydrocarbon chain with optionally adouble bond, optionally having 1-3 oxygen functions, and optionallyforming a bond with a carbon atom in the benzene ring A; R₁, which canbe the same or different, is OH or OCH₃, and n is 0-2; for thepreparation of a medicament inhibiting tyrosine phosphorylation of theinsulin-like growth factor-1 receptor, but not of the insulin receptor:2. Use of a compound having the formula I

wherein the distance between the carbon atom of the methylene group andthe carbon atom of the methoxy group is 0.85-1.05 nm; R is OH, OCH₃,OC₂H₅ or a C₁₋₅ linear or branched hydrocarbon chain with optionally adouble bond, optionally having 1-3 oxygen functions, and optionallyforming a bond with a carbon atom in the benzene ring A; R₁, which canbe the same or different, is OH or OCH₃, and n is 0-2; which compoundinhibits the insulin-like growth factor-1 receptor (IGF-1R), but not theinsulin receptor, for the preparation of a medicament for prophylaxis ortreatment of IGF-1R dependent diseases.
 3. Use according to claim 1 or 2of a compound having the formula I

wherein R is OH, OCH₃, OC₂H₅, CH₃, C₂H₅, or C₂H₄OH; and R₁, which can bethe same or different, is OH or OCH₃, and n is 0-2.
 4. Use according toclaim 1 or 2 of a compound of the formula II

wherein R₂, R₃ and R₄, which can be the same or different, are H, OH, O,OCH₃, or R₂ and R₃ together is a methylenedioxy group, or R₃ and R₄together is an acetonide, carbonate or methylendioxy group; and R₁,which can be the same or different, is OH or OCH₃, and n is 0-2.
 5. Useaccording to claim 1 or 2 of a compound of the formula III

wherein R₂ is H, OH, OCH₃, or OC₂H₅, and R₅ and R₆, which can be thesame or different, are H, OH, OOCH₃, OOCH₂CH₃, OCH₃, or OC₂H₅, or R₅ andR₆ together is an ether or a lactone; and R₁, which can be the same ordifferent, is OH or OCH₃, and n is 0-2; with the proviso that when R₂ isOH this substituent can not be in alpha-position.
 6. Use according toany of claims 1, 2 or 5 of a compound selected from group consisting ofepipodophyllotoxin and deoxypodophyllotoxin.
 7. Use according to claim 1or 2 of the compound acetylpodophyllotoxin.
 8. A compound of the formulaI

wherein R is OH, OCH₃, OC₂H₅, CH₃, C₂H₅, or C₂H₄OH; and R₁, which can bethe same or different, is OH or OCH₃, and n is 0-2 for use as amedicament.
 9. A compound of the formula II

wherein R₂, R₃ and R₄, which can be the same or different, are H, OH, O,OCH₃, or R₂ and R₃ together is a methylenedioxy group, or R₃ and R₄together is an acetonide, carbonate or methylendioxy group; and R₁,which can be the same or different, is OH or OCH₃, and n is 0-2.
 10. Apharmaceutical composition comprising a compound according to claim 8 or9 in combination with a physiologically acceptable carrier.
 11. Useaccording to any of claims 1-7 for the preparation of a medicament fortreatment of cancer.
 12. Use according to claim 11 of a compoundselected from group consisting of epipodophyllotoxin,deoxypodophyllotoxin, and acetylpodophyllotoxin for prophylaxis ortreatment of leukaemia.
 13. Use according to any of claims 1-7 for thepreparation of a medicament for treatment of arteriosclerosis.
 14. Useaccording to any of claims 1-7 for the preparation of a medicament fortreatment of psoriasis.
 15. Use according to any of claims 1-7 for thepreparation of a medicament for treatment of acromegaly.
 16. Use of acompound according to any of claims 1-7 for the preparation of amedicament for treatment of cancer comprising an amount of said compoundeffective to keep the plasma level thereof at a concentration of0.05-5.0 μM.
 17. Method of treatment of a cancer in a mammal, comprisingthe steps of administrating a pharmaceutical composition, containing acompound having the formula I, II or III in combination with aphysiologically acceptable carrier, by constant infusion to a patientsuffering from a tumor, monitoring the plasma level of the compound, andadjusting the rate of infusion to keep the plasma level at aconcentration of 0.05-5.0 μ, for a period of time being sufficient forthe tumor to be retarded or to disappear.